Despite advances in determining the molecular mechanisms regulating both normal and aberrant intestinal growth, colorectal cancer (CRC) remains the 3rd most common cancer in the United States. This merits consideration of other pathways that play important functional roles. MicroRNAs (miRNAs) are endogenous, non-coding RNAs that target mRNAs for transcriptional repression, and are thought to regulate up to 30% of all human genes. Our prior work revealed that members of the let-7 family of miRNAs, which target several mRNAs including c-Myc, K-ras, IMP-1 and HMGA2, are down-regulated in approximately 50% of CRCs. Several recent studies support the emerging paradigm in which the same factors that promote "stemness" in embryonic or adult stem cells may play a critical role in tumorigenesis, and IMP-1 may be such a factor. IMP-1 is a direct target of let-7 and has been shown previously to stabilize c-Myc and IGF-II mRNA, but the mechanisms of IMP-1 in normal and neoplastic intestinal growth remain elusive. Prior studies suggest IMP-1 may have a role in stem cell maintenance or cellular differentiation. Our preliminary data demonstrate that IMP-1 loss decreases proliferation and anchorage-independent growth of CRC cell lines, and that high IMP-1 expression is positively correlated with 2-catenin and K-ras activation in primary CRC tumors. Lin28b is an endogenous repressor of let-7, and cells over-expressing Lin28b display a dramatic increase in IMP-1. In the current proposal, we hypothesize that IMP-1 is a critical regulator of normal intestinal growth and colorectal tumorigenesis as a result of Lin28b-mediated down-regulation of let-7. This hypothesis will be tested through two interrelated specific aims. Aim 1 is to characterize the role of IMP-1 in normal intestinal growth and Lin28b/let-7-mediated colon tumorigenesis in vitro. This will be achieved using IMP-1, Lin28b, and/or let-7 over-expression and silencing systems in cultured organoids from intestinal stem cell reporter mice. The same expression systems will be used to assay proliferation, migration, invasion, and anchorage- independent growth, as well as three-dimensional (3D) organotypic cultures of CRC cell lines. Adding or inhibiting IGF-II in functional assays will test the role of IGF-II in IMP-1-mediated intestinal growth. Aim 2 is to characterize the role of IMP-1 tumorigenesis in vivo. This will be pursued using xenograft mouse models of IMP-1 over-expression, as well as serial transplantation of cells enriched for tumor-initiating properties. Tumor number, size, and morphology will be analyzed, as well as local and circulating IGF-II. IMP-1 xenograft tumors will be monitored serially for growth, invasion, and metastases using bioluminescent and fluorescent imaging, with concurrent histological and biomolecular analysis for effects on IGF-II or other pathways. Taken together, these studies will reveal mechanisms of IMP-1 in normal intestinal epithelial and CRC biology. In addition, the proposed studies will further support the tumor suppressor role of let-7 and underscore the importance of evaluating the CRC therapeutics that mimic the actions of let-7 or inhibit IMP-1. PUBLIC HEALTH RELEVANCE: Colorectal cancer (CRC) is the 3rd most common cancer in the United States, and despite scientific advances in determining the underlying causes, more needs to be done to make CRC curable. The current proposal will use innovative and collaborative approaches to explore a new pathway that is normal during development, but may play a role in promoting CRC growth. Findings from this study will provide evidence for the development of new therapies or biomarkers for CRC.